Phenolic Proﬁle and Antioxidant Capacity of Selected Medicinal and Aromatic Plants: Diversity upon Plant Species and Extraction Technique

: Phenolic content and antioxidant capacity (AC) was evaluated in extracts of bay, sage and thyme leaves, myrtle leaves and berries, and sea buckthorn berries obtained by conventional (CE) and advanced extraction techniques [ultrasound-assisted (UAE) and accelerated solvent extraction (ASE)] using 80% acetone ( v / v ) as extraction solvent. Extracts were analyzed for phenolic content using UPLC/ESI MS 2 and AC by ORAC method. Results indicated the variations in the phenolic composition and concentrations among analyzed plant species and applied extraction methods. Flavonoids showed to be the predominant phenolic group represented by ﬂavonols kaemferol-3- O hexoside (182.58–321.45 mg 100 − 1 g dm) and quercetin-3-glucoside (253.05–315.67 mg/100 g dm) in bay leaves, by ﬂavonol isorhamnetine-3- O -hexoside (27.76–45.16 mg/100 g dm) in sea buckthorn berries and by ﬂavone luteolin-7- O -glucoside (470.27–781.78 mg/100 g dm) in sage leaves. Among the phenolic acids, hydroxybenzoic acids and their derivates were the predominant phenolic group in thyme leaves and myrtle. Statistical analysis showed that ASE contributed to the highest content of total ﬂavonols, ﬂavones, hydroxycinnamic and hydroxybenzoic acids as well as AC. CE was more efﬁcient method for the extraction of total ﬂavan-3-ols, while UAE showed the highest efﬁciency in extraction of total anthocyanins. Analyzed plant extracts proved to be a rich source of various phenolics and results indicated suitable extraction methods for target phenolic compounds characteristic for certain plant species.


Introduction
The medicinal and aromatic plants (MAP) are widely used for culinary purposes to improve the taste and other organoleptic properties as well as in traditional medicine due to their capacity to promote health [1]. Among the numerous MAP, sage (Salvia officinalis L.) and thyme (Thymus vulgaris L.) as powerful members of the Lamiaceae family, bay (Laurus nobilis L.) as a member of Lauraceae family and myrtle (Myrtus communis L.) as a member of Myrtaceae are among the most commonly used plants in traditional Mediterranean remedies. Moreover, sea buckthorn (Hippophae rhamnoides L.), as a member of family Elaegnaceae, is used in oriental traditional medicine mainly for the treatment of lung, skin and gastrointestinal problems and inflammation [2][3][4][5].
These plant species differ in their botanical and morphological characteristics. For example, sage is a perennial, evergreen subshrub, with woody stems, grayish leaves, and blue to purplish flowers. A low-growing hardy perennial, thyme is a fragrant herb with small and oval gray-green leaves, thin, woody stems and tiny pink, tubular flowers. Bay tree is an evergreen with the thick leathery and glossy dark green colored leaves. Myrtle as an evergreen shrub or a small tree is covered by branches, small leaves and starry, scented, white or pink flowers, whereas blue-black berry fruits are small, round shape with seeds inside. Sea buckthorn is a small shrub with a distinct pale silvery-green, lanceolate leaves and orange-yellow globose to egg-shaped berries. Although listed plants have a unique composition, the health benefits they provide originate primarily from bioactive compounds, particularly phenolics and essential oils volatiles. These compounds possess antioxidant, antimicrobial, anti-inflammatory, anticancer, antiviral and antidiabetic effects [6]. Content of phenolics and their molecular structure considerably influence the pharmacological properties of MAP [7]. In particular, the leaves of MAP have been reported to contain higher levels of phenolic compounds and antioxidant activity when compared to berries. However, phenolic content in berries and leaves varies depending on the species, geographical location and the degree of physiological maturity [8,9]. The main phenolic compounds determined in sage and thyme are phenolic acids such as rosmarinic acid, methyl rosmarinate, caffeic acid, cinnamic acid, chlorogenic acid and quinic acid besides flavonoids such as apigenin, luteolin and quercetin [10]. Myrtle leaves and berries are considered as a good source of phenolic acids such as caffeic, ellagic and gallic acid derivates, flavonoids quercetin, catechin, myricetin derivates and anthocyanins in colored berries [11]. The major phenolic compounds found in bay leaves extracts are flavonoids quercetin, kaempferol, rutin, and their derivates as well as phenolic acids such as rosmarinic and caffeic acid, respectively [12]. On the other hand, sea buckthorn berries and leaves are considered to be abundant with flavonol glycosides of isorhamnetin, kaempferol and quercetin derivates [3,4].
Extraction of bioactive compounds is the first step in the analysis of MAP and it plays a crucial role in their further separation and characterization [13]. Currently, there is a considerable rise on the application of MAP extracts as functional ingredients in food and beverage commodities since they present natural, eco-friendly and generally recognized as safe products. Good quality in extract production requires high quality of the raw plant material as well as the appropriate extraction procedure and conditions [14][15][16]. Due to different morphological and botanical characteristics of the above-mentioned MAP, it is important to modify the extraction method in order to obtain higher yields of the target compounds and the lowest possible proportion of ballast substances. In most cases, solid-liquid extraction can be advantageously used for the recovery of natural compounds from untreated or pretreated biomass materials [17,18]. The most commonly used method for the extraction of phenolic compounds from MAP is conventional extraction using heat or stirring and solvents such as water and polar organic solvents (methanol, ethanol or acetone) or their aqueous mixtures [19,20]. Crude phenolic extracts contain complex mixtures of some classes of phenols, which are selectively soluble in the different solvents. According to Dai and Mumper [14], alcohols have been found to be more efficient in extraction of lower molecular weight polyphenols, while acetone is more effective for extraction of higher molecular weight polyphenols. Acetone is a useful extraction solvent, which is highly effective at extracting antioxidants and it is able to dissolve many hydrophilic and lipophilic components. It is widely used for the extraction of tannins and phenolic compounds [16]. According to literature data, acetone showed better extractability of total phenolics from millets grown in India than methanol and water [21] while aqueous acetone (80% v/v) showed as the most effective for the extraction of total flavonoids in baobab [22]. However, advanced extraction techniques have been increasingly explored in recent years in order to improve the extraction efficiency of bioactive compounds and to overcome the disadvantages of conventional extractions such as large solvent consumption, long extraction time and high energy use [20,23]. Some of the most promising methods are ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE), supercritical fluid extraction (SCFE) and accelerated solvent extraction (ASE) [20,[24][25][26][27]. The UAE is based on the principle of cavitation (production, growth and collapse of bubbles in the extraction solvent and sample material), while MAE occurs as the result of changes in the cell structure caused by electromagnetic waves. ASE uses high pressure to keep solvents liquid above their boiling point which facilitates the extractability of the analytes and SCFE employs supercritical fluids as solvent for the extraction of bioactive compounds [14,28,29]. Extraction efficiency of conventional and advanced extraction techniques generally depends on the selection of critical input parameters [29]. The data regarding phenolic content in different plants are difficult to compare since the same extraction conditions can provide different yields of phenolics due to complex nature of sample matrix and differences in plant morphological properties, chemical composition and undesirable ballast content.
Among the broad range of extraction techniques, UAE and ASE have been widely used in the extraction of various phenolic compounds from different parts of MAP without causing significant changes in the structural and functional properties of the target compounds [14,20,25,27]. According to literature data, during UAE temperature and extraction time considerably vary due to sample composition, while solvent type and temperature are the most significant parameters in ASE [30].
Numerous desirable biological effects originating from MAP, such as antioxidant properties, may considerably depend on the amount and composition of isolated phenolic compounds. Phenolic compounds act through several chemical mechanisms as reducing agents, hydrogen donors, singlet oxygen quenchers and metal chelators [31]. Generally, total phenolic content is highly correlated with the antioxidant capacity and leaves have higher antioxidant capacity in comparison with fruits [26,32]. The most frequently used in vitro model for measuring antioxidant capacity in MAP extracts is oxygen radical absorption capacity (ORAC) assay since it is relevant to in vivo conditions [33,34].
Although UAE and ASE of phenolics have been studied for particular MAP species, studies on phenolic extraction from bay, sage and thyme leaves, myrtle leaves and berries, and sea buckthorn berries are scarce. Therefore, the aim of this study was to compare the efficiency of conventional (CE) and advanced extraction methods (UAE and ASE) in the isolation of different classes of phenolic compounds in selected plant species which differ in botanical and morphological characteristics (sage, thyme, bay, myrtle and sea buckthorn). Moreover, biological potential of obtained extracts was also evaluated by determining the antioxidant capacity using ORAC method.

Plant Material
Dry leaves of bay (Laurus nobilis L.), thyme (Thymus vulgaris L.) and sage (Salvia officinalis L.) were obtained from a local manufacturer Suban Ltd. (Strmec Samoborski, Croatia). Dried sea buckthorn (Hippophae rhamnoides L.) berries and leaves and berries of myrtle (Myrtus communis L.) were obtained from a local herbal manufacturer Jerkin (Zadar, Croatia). Botanical identification of plants was performed by the experts from the Faculty of Agriculture, University of Zagreb, Croatia.

Sample Preparation
All plant materials were ground in commercial grinder (GT11, Tefal, France) and sieved through a 60-mesh sieve to obtain uniform particle size. Prior to extraction of phenolics, in each extraction procedure obtained powder (100 g) was defatted using nhexane at 60 • C for 30 min. The solid-liquid mixture was then centrifugated at 4500× g rpm for 15 min (Rotofix 32, HETTICH, Tuttlingen, Germany) and the residue was dried in an oven (FN 500, Nuve, Ankara, Turkey) at 30 • C for 6 h. Dried defatted powders were stored at 4 • C prior to phenolics extraction. The mixture was then centrifuged at 4500× g rpm for 15 min, supernatants were filtered (Whatman No. 4) into 50 mL volumetric flasks, and made up to volume with extraction solvent. Extractions were performed in a duplicate (n = 2) and extracts were stored at −18 • C in nitrogen gas atmosphere until analysis (not longer than 10 days). Prior to UPLC/ESI-MS2 analysis extracts were filtered through 0.45 µm membrane filter (Macherey-Nagel GmbH, Düren, Germany).

ASE
ASE was performed using Dionex™ ASE™ 350 extractor (Thermo Fisher Scientific Inc., Sunnyvale, CA, USA) and the procedure was set according to the method by Repajić et al. [24], slightly modified: 34 mL stainless steel cells fitted with 2 cellulose filters (Dionex™ 350/150 Extraction Cell Filters, Thermo Fisher Scientific Inc.) at the bottom of the cells were filled with a mixture of sample (2.5 ± 0.001 g) and diatomaceous earth (0.5 g). Extraction was conducted using aqueous acetone solution (80%, v/v) as extraction solvent under the temperature 60 • C, static extraction time 5 min, 3 extraction cycles and constant pressure of 10.34 MPa. After extraction, cells were flushed with solvent (50% flushing) and purged with nitrogen for 30 s. Obtained extracts were collected in 250 mL glass vial with Teflon septa, transferred into 50 mL volume flask and made up to volume with the extraction solvent. Extractions were performed in a duplicate (n = 2). Extracts were stored at −18 • C in nitrogen gas atmosphere and analyzed in next 10 days. Prior to UPLC/ESI-MS2 analysis extracts were filtered through 0.45 µm membrane filter (Macherey-Nagel GmbH).

CE
CE procedure included agitation-assisted extraction as follows: 2.5 ± 0.001 g of ground sample was mixed with the 80% aqueous acetone solution (v/v, 40 mL), and the mixture was vigorously stirred for 5 min. Extraction was performed at 60 • C for 30 min, based on our preliminary studies on selected MAP after optimization of process variables such as time (20-120 min) and temperature (30-70 • C) (results not yet published), in water bath shaker (SBS40, Cole-Par-mer, Stone, UK)). The mixture was then centrifuged at 4500× g rpm for 15 min and supernatants were filtered (Whatman No. 4) into 50 mL volumetric flasks, and filled up with extraction solvent. Afterwards, extracts (n = 2) were stored at −18 • C in nitrogen gas atmosphere until analyzed (in a period of 10 days). Extracts were filtered through 0.45 µm membrane filter (Macherey-Nagel GmbH) before UPLC/ESI-MS2 analysis.

Characterization of Phenolic Compounds by UPLC/ESI-MS 2 Analysis
The UPLC/ESI-MS 2 analysis was used for characterization of phenolics using Agilent 1290 RRLC instrument (Agilent, Santa Clara, CA, USA) connected to the Agilent triple quadrupole mass spectrometer (6430 QqQ) with ESI ion source and equipped with binary pump, autosampler and thermostated column compartment. The reverse-phase separations were performed on Agilent Zorbax Eclipse Plus C18 column (100 × 2.1 mm, 1.8 µm particle size) at 35 • C. The solvent composition and the gradient program applied for the analysis was followed as described by Elez Garofulić et al. (2018) [25]. The injection volume of sample was 2.5 µL and the flow rate was set at 0.3 mL min −1 . Electrospray ionization (ESI) was done in both negative and positive ion modes (m/z 100 to 1000) for peak identification and the data were collected in the multiple reactions monitoring mode (MRM). The ionization source parameters for analysis were set as follows: positive/negative capillary voltage, 4000/3500 V, drying gas temperature of 300 • C with a flow rate of 11 L h −1 and nebulizer pressure 40 psi. High purity nitrogen gas (Messer Ltd., Šibice, Croatia) has been used as inducing cone and collision gas. Instrument control, qualitative and quantitative data collection were performed using Agilent Mass Hunter workstation software. Identification of the phenolic compounds was carried out by comparing mass spectra and fragmentation patterns of the authentic standards. Identification of the compounds lacking authentic standards was performed on the basis of previously reported mass spectral data and mass fragmentation patterns [35][36][37][38][39][40][41][42][43][44][45][46][47][48]. All standards were qualified and quantified in MRM mode, using the optimized specific parameters: retention time, precursor ion, product ion, fragmentor voltage, collision energy and ionization mode. Quantitative determinations were carried out using external standard method and in case of lacking analytical standards quantification was carried out using calibration curves of standards from corresponding phenolic group. The limit of detection (LOD) and limit of quantification (LOQ) for the phenolic compounds were expressed by 3-and 10-fold of the ratio of the signal-to-noise (S/N) and reported previously in study Elez Garofulić et al. (2018) [25]. The obtained values of percentage recovery of standard compounds after spiking the sample with the known concentration of standard compounds were from 81-85.83%. Obtained concentrations were expressed as mg per 100 g of dried matter (mg/100 g dm) (n = 2).

Determination of Antioxidant Capacity by ORAC Method
The ORAC assay was conducted on a 96-well microplate using a fluorescence plate reader (Clariostar. BMG LABTECH, Offenburg, Germany) according to the method by Elez Garofulić et al. (2020) [25]. The reaction consisted of 25 µL of diluted extracts and 150 µL of fluorescein (75 nM) which was used as a target for free radical attack. The reaction mixtures were incubated for 30 min at 37 • C and the reaction was initiated by the addition of 25 µL AAPH (240 mM) and the fluorescence was (λ excitation 485 nm i λ emission 538 nm) recorded every 90 sec for 120 min at 37 • C. The blank was sodium phosphate buffer (75 mM, pH 7.4). The Trolox (25 µL) was used as the standard and results were expressed as µmol of Trolox equivalent per g of dried matter (µmol TE/g dm, n = 2).

Statistical Analysis
For statistical analysis Statistica ver. 10.0 software (Statsoft Inc., Tulsa, OK, USA) was applied. Experimental part included full factorial randomized design with two independent factors: (i) plant species-6 levels and (ii) extraction method-3 levels. Descriptive statistics was used for the basic data evaluation. Dependent variables were the contents of total flavonols, flavan-3-ols, flavones, hydroxybenzoic acids, hydroxycinnamic acids and antioxidant capacity. The results were analyzed by multivariate analysis of variance (MANOVA) and marginal mean values were compared with Tukey's HSD test. Significance level p ≤ 0.05 was assigned for all tests.

Results and Discussion
The assessment of the biological potential of extracts of selected plant species was performed on the basis of characterization and determination of mass fractions of phenolic compounds and antioxidant capacity. Same extraction conditions of CE and advanced extraction techniques (UAE and ASE) were used to compare their efficiency. Characterization of phenolic composition in bay (Laurus nobilis L.) leaves, sage (Salvia officinalis L.) leaves, thyme (Thymus vulgaris L.) leaves, myrtle (Myrtus communis L.) leaves and berries and sea buckthorn (Hippophae rhamnoides L.) berries was analyzed by UPLC/ESI-MS 2 method.  Table 1). With regard to botanical and morphological characteristics, differences in the phenolic composition of plants were observed and the identified compounds are arranged into their constituent groups and occurrence in each of the MAP extracts (Table 2).
In current study, isorhamnetin-3-rutinoside and isorhamnetin-3-rhamnoside were determined only in sea buckthorn berries (4.12-22.05 mg/100 g dm) which contents are in accordance with research of Pop et al. (2013) [43]. The isoramnethin-3-O-hexoside was present in analyzed extracts of sage, thyme and bay leaves and sea buckthorn berries. The highest concentration of isoramnethin-3-O-hexoside was determined in bay leaves extracts (67.18-96.09 mg/100 g dm). Similar results were obtained in the study of Dias et al. (2014) [49] where concentration of isoramnethin-3-O-hexoside in cultivated and wild bay leaves ranged from 0.2 to 1.29 mg/g of extract.
Myricetin and glycoside derivates were the major flavonoid constituents determined in myrtle leaves and berries. Cp 11-13 were tentatively assigned as myricetin derivates due to characteristic fragment ion at m/z 319.
Belonging to the class of FLAOL, cp 19, 20 and 21 were identified by comparison with authentic standards and were assigned as catechin, epicatechin and epigallocatechin gallate. Cp 22 (m/z 305) was assigned as gallocatechin on the basis of fragmentation pattern with the fragment ion m/z 225 [39].
Among identified FLAOL the most abundant compounds were epicatechin (56.01-82.44 mg/100 g dm) determined in bay leaves and epigallocatechin gallate (47.73-64.52 mg/100 g dm) determined in myrtle leaves, respectively. Dias et al. [49] reported that FLAOL were the major phenolic compounds present in bay leaves with (-)-epicatechin (12.35-23.08 mg/g) being the most abundant one. Romani et al. (1999) [36] reported higher content of epigallocatechin in myrtle leaves (2710 µg/g dm) in comparison with results in current study.
In addition to flavonoids, analyzed plant extracts contained a large amount of phenolic acids (HBA and HCA) and LG.
Cp 29, 34-36 and 38-40 were identified by comparison with authentic standards as gallic, vanillic, caffeic, chlorogenic, ferulic, p-coumaric and rosmarinic acid. The ESI-MS signals at m/z 153 and 137 were identified as protocatechuic and 4-hydroxybenzoic acids on the basis of fragmentation pattern with the fragment ions m/z 109 and 93 which correspond to the loss of CO 2 from their precursor ions, respectively [39].
Cp 31 and 32 determined in both myrtle leaves and berries were assigned as gallic acid derivates tentatively identified as digalloylquinic acid and 5-O-galloylquinic acid due to precursor ions at m/z 495 and 343 and fragment ions at m/z 343, 191, 169 and 125 [41,44,61].
The most abundant HBA was 5-O-galloylquinic acid determined in myrtle leaves (722.01-872.43 mg/100 g dm) and berries (369.43-551.93 mg/100 g dm). A considerably lower content of gallic acid and galloyl derivates found in myrtle leaves extracts was reported by Taamalli  Cp 41 and 42 determined in sage with MS 2 fragment at m/z 359 corresponding to rosmarinic acid were identified as salvianolic and sagerinic acid. The fragmentation pattern was previously reported by Sharma et al. (2020) [48]. The presence of caffeic and rosmarinic acid and its derivates was previously reported by Lu and Foo (2000) [57] and Hossain et al. (2010) [39]. The major phenolic acids in Salvia sp. are caffeic acid derivates (Kamatou, 2010) [64]. Among the identified HCA, rosmarinic acid was detected in the highest amount in sage extracts (257.63-496.42 mg/100 g dm). These results are in accordance with the study of Martins et al. (2015) [53] where rosmarinic acid was the most abundant phenolic acid in extracts of sage.
Current study showed great diversity and complexity of natural mixtures of phenolic compounds in extracts of morphologically and botanically different plants. The total content of phenolic groups determined in selected MAP extracts of leaves and fruits obtained with different extraction techniques are shown in Table 2. Results showed that dominant group of phenolic compounds were FON in sage, FOL in bay and sea buckthorn, and HBA in thyme and myrtle.
When observing the influence of plant species on the content of phenolics, the difference in total content of each phenolic group was statistically significant (Table 3). In general, the highest content of total FON (806.66 mg/100 g dm), HCA (436.98 mg/100 g dm) and LG (298.91 mg/100 g dm) was determined in extracts of sage leaves. Total FLAOL (84.99 mg/100 g dm) and HBA (799.61 mg/100 g dm) were the highest in extracts of myrtle leaves, while the highest content of total FOL (866.31 mg/100 g dm) characterized extracts of bay leaves.
The content of phenolic compounds in selected MAPs also varied upon extraction method used ( Table 2). The highest contents of total FOL and FON in sage, thyme, bay, myrtle and sea buckthorn extracts were obtained when ASE was applied, except for FON in myrtle berries which were the highest in extracts obtained by CE. ASE also contributed to the highest content of total HBA in extracts of leaves of sage, thyme and bay, and myrtle berries. Moreover, the highest yields of total HCA in sage leaves and myrtle berries extracts were also achieved when ASE was used. This good performance of ASE derives from the fact that under high pressure solvent remains in liquid state even at temperatures higher than their boiling point which improves the extraction efficiency by "pushing" the solvent into pores and allowing better penetration into the matrix [70]. Rapid solvent penetration prevents the degradation of phenolic compounds despite that ASE is carried out under high temperature and pressure [71]. Contrary, ASE was the least suitable method for the extraction of total HBA in sea buckthorn berries and total FLAOL, HBA and HCA in myrtle leaves. The UAE was more efficient in comparison with ASE and CE in extracting FLAOL from sea buckthorn, HCA from thyme and sea buckthorn, and ANT from myrtle berries. On the other hand, CE contributed to the higher content of FLAOL, HBA and HCA in extracts of myrtle leaves, LG in sage and thyme, FLAOL in bay and HBA in sea buckthorn. UAE is useful method for thermolabile compounds since active components rapidly diffuse by sonication from plant tissue to solvent, thus shortening the extraction time [72]. The choice of an appropriate extraction method considerably depends on the nature of the plant matrix [71]. For example, literature data showed that ASE was more suitable extraction method for mulberry, UAE for myrtle berries and CE for sweet purple potatoes phenolics [73][74][75].
Various structures of phenolics are present in different MAP and they have different physical and chemical characteristics (molecular structure, polarity, concentration, number of aromatic rings and hydroxyl groups) as well as diverse interactions with other compounds (proteins or carbohydrates). This significantly affects the extraction processes [14]. Therefore, it is difficult to use single extraction method for all classes of MAP phenolic compounds. Still, advanced extraction techniques are always preferred due to great disadvantages of conventional extraction procedures. Analyzed extracts of selected MAP showed to be a good source of different phenolics and obtained results indicated suitable extraction methods for target phenolic compounds characteristic for certain plant species.
Regarding extraction methods, sage, bay, sea buckthorn and myrtle berries extracts obtained by ASE and extracts of myrtle and thyme leaves obtained by CE showed the highest ORAC values. ORAC values determined in plant extracts of myrtle and sage were in accordance with previously reported data [76,77]. Moreover, literature data suggested that myrtle extracts have a potent AC mainly due to presence of HBA, FOL and galloyl derivatives [61,78]. In study of Kratchanova et al. (2010) [79], higher AC was determined in extracts of thyme (1434-1637 µmol TE/g) and bay (170-837 µmol TE/g) when compared to AC values in current study. Higher AC in sea buckthorn (18.41-34.68 mmol TE/100 g) was also determined in study of Tkacz et al. (2019) [80].
Based on statistical analysis, significant differences in ORAC values between analyzed plant extracts were observed (Table 3). Sage extract showed the highest ORAC level (859.23 µmol TE/g dm), while the lowest ORAC value described sea buckthorn extract (70.74 µmol TE/g dm). According to Lu and Foo (2001) [81] rosmarinic acid derivatives considerably contribute to AC of sage. Furthermore, applied extraction method also significantly affected AC (Table 3). It can be seen that ASE contributed to the highest AC in plant extracts (535.11 µmol TE/g dm), while lower AC was observed in plant extracts obtained by CE and UAE. Higher AC was also reported in mulberry extracts obtained by ASE when compared to UAE [73].
It can be concluded that all analyzed plant extracts showed potent AC and differences in AC among plant extracts may be attributed to the content of phenolic compounds present in the extract as well as the extraction method applied.

Conclusions
Significant variability in the phenolic composition among extracts of selected plant species [bay (Laurus nobilis L.), sage (Salvia officinalis L.) and thyme leaves (Thymus vulgaris L.), myrtle leaves and berries (Myrtus communis L.) and sea buckthorn berries (Hippophae rhamnoides L.)] was observed. The flavonoids such as FOL, FLAOL, FON, ANT and LG were the predominant phenolic groups in bay, sage and sea buckthorn, while HBA and HCA and their derivates were the most abundant in thyme and myrtle extracts. Results revealed that the most represented compounds in bay extracts were kaempferol-3-O-hexoside and quercetin-3-glucoside, while isorhamnetine-3-O-hexoside and luteolin-7-O-glucoside were the most abundant in sea buckthorn and sage extracts, respectively. In thyme and myrtle extracts HBA and its derivates were represented by 4-hydroxybenzoic and 5-Ogalloyl-quinic acid. Moreover, yields of phenolics were strongly influenced by extraction technique used. ASE showed to be the most appropriate extraction method for FOL, FON, HCA and HBA, while UAE and CE showed better efficiency in extraction of ANT and FLAOL, respectively. The results of current study proved that sage, myrtle and bay extracts obtained by ASE contain different phenolics and high AC levels and therefore possess a great potential to be used as a source of natural antioxidants in production of functional and added-value products.